Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Protocol: ATCCNO: 50177 SPEC: The following directions for recovery from the frozen preparation must be carefully followed if a culture is to be successfully established: 1. Upon receipt, it is necessary to store the frozen ampule at temperatures of -135C or lower for at least 48 hours prior to thawing. The ampule may be stored in a mechanical refrigerator set at -60 to -70C if the storage time does not exceed 48 hours. The ampule should be removed from the dry-ice when stored in either a mechanical or nitrogen refrigerator. 2. After the frozen ampule is placed in storage, prepare a single tube of reduced ATCC medium 1671 in the manner outlined in step 1 under Maintenance of Strain and Subculturing below. 3. When the medium is ready for inoculation, thaw the ampule in a 35C water bath without agitation (do not fully immerse ampule) and remove vial immediately after the preparation has become completely liquid. 4. Aseptically, gently lower a sterile Pasteur pipette from which the air has been expelled to the bottom of the liquid in the ampule and slowly aspirate the entire contents into the pipette. Be careful to minimize agitation of the fluid and do not introduce air bubbles at any time. These organisms are sensitive to oxygen. 5. Add the cell suspension to the bottom of the liquid overlay of the reduced tube of ATCC medium 1671. Avoid expulsion of air bubbles from the tip of the pipette. 6. With the cap of the test tube loosened (1 full turn) place it in an anaerobic jar which contains a BBL GasPak (one anaerobic system GasPak per BBL GasPak 100 anaerobic culture jar). Close the vessel securely and incubate at 35C. Maintenance of Strain and Subculturing: Subculture at 2-3 day intervals. 1. Prior to inoculation, reduce (lower redox potential) complete ATCC medium 1671 by placing the tubes with caps on loosely (1 full turn) in an anaerobic jar containing a BBL GasPak (one anaerobic system GasPak per BBL GasPak 100 anaerobic culture jar) for at least 48 hours. Note: The palladium catalyst in the GasPak jar should be replaced biweekly with fresh catalyst. 2. To subculture, remove the growing cultures without agitation from the anaerobic jar and immediately screw the caps down tightly. The strain grows at the bottom of the liquid overlay as a dense mass of cells. 3. Carefully introduce a sterile Pasteur pipette aseptically through the liquid overlay - air interface (avoid expulsion of air bubbles) and move the tip of the pipette to the cell mass, aspirate approximately one third of the mass into the pipette. Tighten the cap immediately unless the culture is to be promptly placed in the anaerobic jar. 4. Place the freshly inoculated culture into the anaerobic jar with the cap loosened (1 full turn), prepare the GasPak and quickly seal the jar.

Cryopreservation

1. Two to three days in advance, prepare fresh tubes containing ATCC medium 1671 and 25% HIHS in an anaerobic jar with tube caps loosened one full turn. Add a BD GasPak (one anaerobic system GasPak per anaerobic culture jar). Close the vessel securely and incubate at 35°C for at least 48 hours. If the GasPak is of the variety that makes use of a palladium catalyst, the catalyst should be replaced biweekly.

2. Using the liquid overlay from the previously-reduced tubes of medium prepared in step 1 above, prepare a 20% (v/v) sterile DMSO solution in Stone's Modification of Locke's Solution in the following manner:

b) Place the tube on ice and allow the DMSO to solidify (~5 min), then add the required volume of previously-reduced liquid overlay. Chemical heat will be liberated from this combination so allow the solution to cool to room temperature;

c) If time allows, loosen the tube cap one full turn and place in an anaerobic jar with an anaerobic GasPak for at least 48 hours prior to use.

3. When the test tube cultures are at or near peak density remove the tubes from the anaerobic jar and immediately screw the caps on tightly. Opening one tube at a time, gently remove the cells from the bottom of the egg medium slants and pool in a single 16 x 125 mm screw-capped test tube (work quickly to avoid prolonged exposure to air).

4. Adjust the cell concentration to 1.0-2.0 x 107cells/ml using overlay from a reduced tube of medium. If the concentration of cells is too low centrifuge at 500 x g for 5 minutes. Adjust the volume of supernatant to yield the desired final cell concentration.

5. Mix the cell preparation and the cryoprotective agent prepared in step 2 in equal portions. Thus, the final concentration will equal 10% (v/v) DMSO and 5.0 x 106 - 1.0 x 107 cells/ml. The time from the mixing of the cell preparation and glycerol/DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.

7. Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately

-1°C/min.)

8. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -55°C.

9. Before thawing an ampule do the following: Place tubes containing ATCC medium 1671 and 25% HIHS in an anaerobic jar with tube caps loosened one full turn. Add a BD GasPak (one anaerobic system GasPak per anaerobic culture jar). Close the vessel securely and incubate at 35°C for at least 48 hours. If the GasPak is of the variety that makes use of a palladium catalyst, the catalyst should be replaced biweekly.

10. Thaw the frozen ampule in a 35°C water bath without agitation until all of the contents are liquid (about 2-3 minutes).

11. Aseptically and gently, lower a sterile Pasteur pipette from which the air has been expelled to the bottom of the liquid in the ampule and slowly aspirate the entire contents into the pipette. Be careful to minimize agitation of the fluid and so not introduce air bubbles from the tip of the pipette.

12. Inoculate a fresh tube of previously-reduced ATCC medium 1671 by inserting the Pasteur pipette tip aseptically through the liquid overlay-air interface (avoid expulsion of air bubbles or culture) and moving the tip of the pipette to the base of the solid-overlay interface. Expel the entire contents of the Pasteur pipette into the culture tube (again avoid expulsion of air bubbles), then tighten the cap immediately unless placing the tube directly into an anaerobic jar.

13. With the cap of the freshly inoculated test tube loosened one full turn, place it in an anaerobic jar containing a BD GasPak and incubate at 35°C.

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.